MHC class II molecules present peptides derived from self and foreign antigens to CD4 T cells. Peptides from exogenous and endosomal antigens, are captured by class II molecules for display on the cell surface. Remarkably, epitopes from endogenous cytoplasmic and nuclear antigens also gain access to class II molecules. The purpose of this project is to elucidate the pathways required for processing and delivery of cytoplasmic and nuclear Ag to class II proteins. Our studies suggest cytosolic proteases cleave select cytoplasmic antigens prior to peptide transport by chaperone mediated autophagy (CMA) to class II molecules resident in the endosomal network. Yet, nuclear and cytoplasmic antigens may also be engulfed into autophagosomes via macroautophagy, for delivery to class II molecules. Studies are proposed to dissect the role of specific heat shock proteins and lysosomal membrane proteins in targeting epitopes for class II endogenous antigen presentation. We hypothesize that CMA controls the presentation of select cytoplasmic Ag while macroautophagy randomly sequesters intact nuclear and cytoplasmic Ag for class II presentation. In aim 1, experiments will elucidate the mechanisms by which heat shock protein family members regulate autophagy and class II presentation. Aim 2 will investigate the role of lamp-2 protein isoforms in controlling class II presentation of antigens via autophagy as well as exogenous delivery into antigen presenting cells. In aim 3, studies are proposed to localize compartments within the endosomal network where peptides derived from cytoplasmic and nuclear Ag intersect class II molecules. Addressing these issues will offer critical insights into class II cytoplasmic and nuclear Ag presentation, and the establishment of CD4 T cell responses to autoantigens and intracellular pathogens. This work has relevance to the development of inflammatory autoimmune disorders, as well as the induction of self tolerance to endogenous antigens. Pathways such as CMA often appear to target misfolded intracellular antigens, increasing the potential for reactivity to altered self. Proposed tests of small molecular mass compounds which regulate protein folding and autophagy, may lead to new approaches to prevent autoimmunity and inflammation. Studies of these novel class II pathways may also be useful in vaccine development for infectious agents and tumors.